The present invention relates to the active transfer of haptens, proteins, nucleic acids and other molecules into the nucleus of eucaryotic cells. This invention is of major importance since it can be applied to various fields, especially those of gene therapy and vaccines.
Gene therapy continues to be dependent on a considerable number of parameters among which are the development of vectors capable of transporting through the cytoplasm of these cells of the host organism active principles endowed with predetermined specific properties into the nuclei of cells of the organism in the absence of genetic alterations associated with the use of these vectors, and the non-degradation of the biological activity of the active principles transferred. It is known that so far all these conditions are far from being fulfilled (1).
Indeed, the current methods commonly used to transfer DNA into cells are the following: general, non-selective methods which use the property of DNA to coprecipitate with calcium phosphate or DEAE-dextran, or alternatively the direct introduction of DNA into cells under the effect of an electric field (electroporation). These methods are very toxic for the cells, leading to a high mortality and a high variability according to the cells used. Other methods use targeting of the entry of the gene into cells by receptors present on their membrane. The DNA may then penetrate into the cell via either a ligand specific for these receptors: asialorosomucoid (2), insulin (3) or transferrin (4), or antibodies specific for membrane constituents (5). The DNA/ligand complex penetrates into the cell by a process of endocytosis. The transfection is therefore limited by a substantial destruction of the complex in the lysosomal vesicles, and different methods have been proposed to overcome these disadvantages, especially the blocking of the lysosomal compartment by chloroquine or the simultaneous addition of adenoviruses which escape the endosomal compartment by destroying the membrane of the endocytosis vesicles (6).
The aim of the present invention is to provide a new type of vectors which are both more efficient and safer than the viral vectors whose use has been envisaged until now.
The invention therefore relates to a product of coupling between a biologically active principle and one of these new vectors, hereinafter called xe2x80x9cimmunovectorsxe2x80x9d, the said product of coupling being characterized both by the capacity of the immunovector to allow the internalization in eucaryotic cells of biologically active principles linked covalently or non-covalently to these immunovectors, and by their affinity for the DNA of these cells to such a point that the said immunovector is rendered capable of transferring the biologically active principle immediately close to the nuclei of these cells or into the nuclei of these cells.
These immunovectors preferably consist of antibodies or fragments of antibodies capable of recognizing DNA sequences inside these cells, and to which biologically active principles may be covalently or non-covalently linked, these antibodies or fragments of antibodies being, in addition, capable of transporting in vitro and in vivo these biologically active principles through the membranes and the cytoplasm of these cells, and transferring them close to or even into the nucleus of these cells.
It is understood that in the present description the term xe2x80x9cbiologically active principlexe2x80x9d relates to any molecule, macromolecule or group of molecules having biological activity of the type in question.
The invention also relates to a method of transferring especially haptens, proteins and/or nucleic acids into the nucleus of cells, particularly eucaryotic cells, this method being based on the use of the properties of the said immunovectors.
The existence of antibodies capable of penetrating inside the nuclei of human lymphocytes when these cells are incubated in vitro in a culture medium containing a serum obtained from patients suffering from disseminated erythematous lupus (DLE) was reported for the first time by Alarcon-Segovia et al. in 1978 (7). Subsequently, the same team demonstrated that these antibodies are of the IgG isotype and are capable of reacting with ribonucleic acids, free or complexed with proteins (8). Recently, this type of antibody was detected in MRL lpr/lpr lupus mice, but also in NZB mice having a haemolytic auto-immune disease syndrome and even in normal BALB/c mice. Some monoclonal antibodies, prepared from the spleen of these mice, have proved capable of penetrating in vitro into the nucleus of cells maintained in culture (10-13). As in humans, it was noted that these monoclonal antibodies were capable of recognizing nucleic acids. Furthermore, it was shown that these antibodies are also capable, when they are injected into mice, of penetrating into several types of cells, ending up in their nuclei (11).
The invention results from the discovery that this type of antibody or fragments of these antibodies could also be used as vectors, hereinafter xe2x80x9cimmunovectorsxe2x80x9d capable of transporting biologically active principles, such as haptens, proteins, and nucleic acids through the membranes and the cytoplasm of the corresponding cells, and ensuring their transfer into the nucleus of the said cells.
These antibodies may be obtained in polyclonal form from a serum, in particular from an animal previously immunized against nucleic acid fragments having the corresponding epitope, or in monoclonal form from hybridomas secreting such antibodies.
Any type of bonding, chemical or otherwise, may be used to ensure the coupling of an immunovector of antibody or antibody fragment type having an affinity for the nucleic acids to the biologically active principle, for example a hapten or a nucleic acid, for the purpose of transporting it through the membranes and the cytoplasm of the cells, and to ensure the transfer of these active principles into the nucleus.
Preferably, a chemical mode of coupling, allowing the formation of covalent or non-covalent bonds, will be used.
Preferred coupling products are those in which the immunovectors are selectable by a cellular penetration test comprising a first incubation of the immunovector of interest in the presence of cells in culture in the nucleus of which the active principle capable of being associated with the immunovector has to be transported, followed, after fixing and permeabilization of these cells, by another incubation with labelled anti-immunovector antibodies, and finally by a detection immediately close to the nucleus or even inside the nucleus of the antigen-antibody type immunologic reaction between the immunovector and the anti-immunovector antibody.
Among the preferred immunovectors of the present invention, there may be mentioned the antibodies having an affinity for a nucleic acid, or a fragment thereof, the latter retaining this affinity.
Antibodies also having the capacity to bind to the cells, in particular lymphoid cells, can also be used. The latter category of immunovectors may also be selected by a test, which may then also comprise the incubation of the immunovectors of interest with lymphoid cells, washing the said lymphoid cells, incubating them with labelled anti-immunovector antibodies, and determining the number of positive cells in each population.
In one embodiment, the lymphoid cells used are auto-immune mouse splenocytes exhibiting a lupus syndrome.
A preferred immunovector for the coupling product is chosen from among monoclonal IgG""s, (Fabxe2x80x2)2 or (Fabxe2x80x2) fragments, or any polypeptide corresponding to the site(s) of the antibodies involved in the recognition of the corresponding nucleic acid.
Preferably, this immunovector is an immunoglobulin, more particularly an IgG carrying an anti-DNA activity, and obtained from normal individuals.
In addition, this immunovector may be an IgG carrying an anti-DNA activity and obtained from individuals presenting auto-immune syndromes, more particularly disseminated erythematous lupus syndromes.
In a specific embodiment of the invention, the immunovector coupled to the active principle is a bi-specific antibody recognizing on the one hand the DNA, and on the other hand a protein such as Tat, Rev of the HIV retrovirus, as well as surface markers such as CD3, CD4, CD8, CD19 and CD34.
Preferably, the biologically active principle coupled to the immunovector is a molecule selected from especially nucleic acids, proteins especially enzymes, for example peroxidase, haptens especially biotin or fluorescein, enzyme activators or inhibitors and medicaments.
In a preferred embodiment, the coupled nucleic acid is a polynucleotide, the immunovector being an IgG, with the coupling being carried out via p-benzoquinone at the rate of one molecule of immunovector per 4 molecules of polynucleotide.
A biologically active principle preferably used consists of a plasmid intended to integrate into the nucleus of the target cells for the expression of a protein encoded by a gene contained in the said plasmid. When such a plasmid is coupled to an immunovector of the antibody type, having affinity for DNA, this immunovector is, preferably, previously coupled to an agent capable of inducing a compacting effect on the DNA, this agent being preferably polylysine.
Indeed, polylysine, which by virtue of its cationic properties is capable of compacting DNA, promotes the transfection of cells. The coupling of polylysine to the immunovector, which is carried out with the aid of a coupling agent, more particularly a carbodiimide such as EDC (1-(3-dimethylaminopropyl)-1xe2x80x2-ethylcarbodiimide), allows the DNA to react therewith. This has the effect of inducing the liberation of the active site of the antibody which may sometimes be masked when the antibody is coupled to an active principle of the size of a plasmid.
Other agents capable of having, by virtue of their cationic properties, a compacting effect on DNA (2-6) may also be coupled to the immunovector to fulfil such a function.
More specifically, a biologically active principle preferably used consists of a gene intended to integrate in the genome of target cells, especially by homologous recombination, more particularly a xe2x80x9cnudexe2x80x9d DNA containing a nucleic acid sequence coding for a polypeptide originating from bacterial or eucaryotic cells, fungal cells or viruses, this polypeptide having vaccinating properties.
Advantageously, this active principle allows the immortalization of selected types of cells, particularly macrophages, dendritic cells, B and T cells, and hematopoietic cells, especially of human origin.
Still more preferably, this biologically active principle is an antisense oligonucleotide allowing the inhibition of protein or nucleotide synthesis, for example in cells infectable by an HIV retrovirus, or in tumour cells.
Thus, the inventors have, on the one hand, tried to obtain, from the spleen of autoimmune mice (NZBxc3x97NZW)F1, having a lupus syndrome, IgG monoclonal antibodies which have been selected for their capacity to react with DNA but also for their capacity to penetrate as far as the nucleus of the cells. In parallel, polyclonal antibodies reacting with the DNA and capable of penetrating into the nuclei of the cells have been isolated by affinity chromatography. This chromatography was applied either to a xe2x80x9cpoolxe2x80x9d of sera of normal patients or to a normal individual serum, preferably to sera obtained from patients suffering from infections, particularly to a serum of normal patients suffering from DLE, or to a mouse (NZBxc3x97NZW)F1 serum.
In a method of selection of immunovectors according to the present invention, the test of cellular penetration of the immunovectors comprises a first incubation of eucaryotic cell lines in a medium comprising the said immunovectors preferably in increasing concentration, then the fixing, and, if necessary, the permeabilization, or vice versa, of these cells, followed by an incubation of the said cell lines with anti-immunovector antibodies preferably labelled with fluorescein or with peroxidase, and the localization of the antibodies thus labelled close to the nuclei of the said cells or better still inside the nuclei. The cell lines are especially chosen from among fibroblasts, thymocytes or splenocytes.
Without the following reaction conditions having a limiting character, it may be mentioned that the first incubation is often carried out at 37xc2x0 C., for about 2 to 8 hours with immunovector concentrations of about 1 to 70 xcexcg/ml, on cell lines inoculated at a concentration ranging from 5xc3x97103 to 5xc3x97106 cells per milliliter.
In one of the embodiments of the method, the cell line is a fibroblast line in exponential growth inoculated at a concentration of 2xc3x97104 cells per milliliter, or thymocytes or splenocytes of BALB/c mice, which are suspended at the rate of about 106 cells per milliter.
Moreover, the invention relates to a method of preparation of immunovector-molecule(s) coupling product, the immunovectors being chosen from among antibodies, more particularly the IgG""s obtained according to the method of selection, (Fabxe2x80x2)2 or (Fabxe2x80x2) fragments, or any polypeptide corresponding to the site of the antibodies or fragments of antibodies involved in the transport of the molecules.
In the method of preparation of a coupling product according to the invention, it is ensured that for each immunovector is coupled at least one molecule of biologically active product, the said molecule being preferably covalently linked to the immunovector.
The following examples illustrate conditions in which haptens such as fluorescein and biotin, small molecules such as hormones, proteins preferably enzymes, enzyme inhibitors or activators and medicines, for example antivirals such as acyclovir or AZT, may be actively transported through the cytoplasm of the treated cells and transferred into the nucleus of the said cells. In particular, fluorescein has been coupled to the free amino groups of the immunovectors via an active isothiocyanate group and the biotin via an active succinimide ester. The coupling of the haptens and the like to the immunovector may be carried out by means of other homo- or heterobifunctional bridging groups or reagents known in the literature, such as the imido esters and N-hydroxysuccinimidyl esters which are capable of reacting with the amino groups, for example derivatives of alkyl, haloaryl, haloacetyl and pyridyl disulphide groups reacting preferentially, maleimides, or by means of sulphydryl groups, carbodiimides, as well as molecules having photoactivable groups such as azidobenzoyl hydrazide (13).
In addition, the inventors have prepared products of coupling where the immunovector is a bi-specific antibody against a target antigen, constructed either by the chemical route or by the genetic engineering route, and have in parallel immunized individuals, for example mice, with the said target antigens, and selected immunovectors which are bi-specific antibodies preferably reacting with the said target antigens, such that the action of the immunovectors is directed specifically.
Techniques relating to the synthesis of bi-specific antibodies have especially been described by Porstmann et al. in 1984 (16), a study entitled xe2x80x9cDevelopment of a bispecific monoclonal antibody for use in molecular hybridisationxe2x80x9d having moreover been published in 1984 by Auriol et al. (17).
Advantageously, the bi-specific antibodies used in this method recognize, inter alia, the proteins Tat, Rev of the HIV retrovirus, as well as surface markers CD3, CD4, CD8, CD19 and CD34.
Moreover, the present invention relates to a method for transferring an active principle in the nuclei of selected eucaryotic cells, characterized by coupling this active principle with an immunovector having both the capacity of allowing internalization of this active principle in these eucaryotic cells and an affinity for the DNA of these cells to such a point that the said immunovector is rendered capable of transporting this biologically active principle immediately close to or into the nuclei of these cells.
This biologically active principle may be covalently or non-covalently coupled to the immunovector.
Preferably, this method of transfer is characterized in that the immunovector entering into the composition of this product is selected from those which are selectable by a cellular penetration test comprising a first incubation of the immunovector of interest in the presence of cells in the nucleus of which the active principle capable of being associated with the immunovector has to be transported, followed, after fixing and permeabilization of these cells, by another incubation with labelled anti-immunovector antibodies, and finally the detection immediately close to the nucleus or even in the nucleus of the antigen-antibody type immunologic reaction between the immunovector and the anti-immunovector antibody.
In a preferred embodiment of the method of transfer according to the invention, the immunovector used is formed of an antibody having an affinity for a nucleic acid, or of a fragment of this antibody retaining this affinity.
This immunovector used in this method is preferably selected from among antibodies, preferably monoclonal IgG""s, (Fabxe2x80x2)2 or (Fabxe2x80x2) fragments, or any polypeptide corresponding to the site(s) of the antibodies involved in the recognition of the corresponding nucleic acid.
Advantageously, once the product of immunovector/active principle coupling is prepared, it may be used for the intranuclear transfer of other molecules. Thus, the fluorescein/immunovector conjugate tested may be associated with an anti-fluorescein antibody coupled with a third molecule, and thus transfer the said third molecule into the nuclei of the cells. Similarly, the biotin/immunovector conjugate may allow the binding of an anti-biotin antibody or of avidin-streptavidin coupled with a third molecule to be transferred into the nuclei.
In the present invention, an enzyme such as horseradish peroxidase was transferred into the nucleus, but other proteins having varied biological activities may also be used. Peroxidase coupled to the immunovector via glutaraldehyde has also been used. However, other methods known in the literature, such as those described in the case of haptens, may also be used.
As in the case of the hapten/immunovector conjugates, there may be used, in association with the protein/immunovector conjugates, an anti-protein antibody coupled with a third molecule for the intranuclear transfer of the said molecule.
In the invention described herein, although a polynucleotide has been transferred into the nucleus, a wide variety of nucleic acids having appropriate biological activities may also be actively transferred at the intranuclear level.
Thus, a method of transfer of active principles according to the invention allows in particular the transfer of genes intended to integrate into the genome of the target cells, especially by homologous recombination, more particularly the transfer of xe2x80x9cnudexe2x80x9d DNA, it being possible for the latter especially to be used as xe2x80x9cDNA vaccinexe2x80x9d.
One of the homologous recombination techniques possible is that described by Mouellic et al. in 1990 (18).
Recent studies carried out by Whalen R. G. et al. have made it possible to show the existence of an immune response following a DNA transfer. These studies, which have been the subject of patent application WO 95/11307, were more particularly applied to the expression of monoclonal molecules of the IL2 cytokine type (19).
In addition, this method of transfer makes it possible to introduce nucleotide sequences involved in the immortalization of different cell types, particularly macrophages, dendritic cells, B and T cells, and haemotopoietic cells especially of human origin. As nucleotide sequences, there may be mentioned the oncogenic sequences or viral sequences associated with cell transformation phenomena.
It also allows the transfer of antisense oligonucleotides allowing the inhibition of protein or nucleotide synthesis, for example in cells infectible by a retrovirus such as HIV, or tumour cells.
In the present invention, the polynucleotide was coupled to the immunovector via p-benzoquinone. However, other methods known in the literature may also be used.
Moreover, the present invention also relates to the eucaryotic cells containing active molecules preferably at the nuclear level, characterized in that the said molecules cannot be naturally incorporated into the nuclei of the said cells or have a weak expression level in the said cells. These molecules are presented in these cells immediately close to their nuclei or into their nuclei, and are coupled to an immunovector characterized by its affinity for the DNA of these cells, in the form of a coupling product according to the invention.
Among the cells to which the present invention relates, there are especially the cells which can be infected by a virus or tumour cells.
Also entering within the framework of the present invention are the hybridomas producing the antibodies according to the present invention, as deposited at the CNCM on Jun. 30, 1995 under the numbers I-1605, I-1606 and I-1607.
In addition, the invention relates to a pharmaceutical composition, characterized in that it contains, in combination with a physiologically acceptable vehicle, a coupling product according to the invention in which the biologically active principle is a medicine or vaccinal active principle and the immunovector is compatible with the host organism to which the medicine is directed.
Also entering into the framework of the present invention is the use of the coupling product of the invention for the expression, in receiving cells, of a nucleotide sequence which is heterologous to the DNA of the host.